Detection of Synaptic Proteins with Fluorescent Molecular Rotor-labeled Peptides Abstract Our goal is to establish routine methodology for development of synthetic peptides for instant and specific detection of endogenous unmodified proteins in fixed and living neurons. We will screen One Bead One Compound (OBOC) combinatorial peptide libraries invented by co-investigator (Co-I) Kit Lam (Nature 354, 82- 84) for peptides with fluorescent molecular rotors (FMRs). FMR peptides will fluoresce only when specifically bound to their target protein but not when free in solution. Initial focus will be on the key postsynaptic proteins of glutamatergic synapses (AMPARs (AMPA-type glutamate receptors), NMDARs (NMDA-type glutamate receptors), PSD-95 (anchors AMPARs and NMDARs at postsynaptic sites)). Signal to noise ratio (>2300fold) and photostability (by ~ 10fold) is superior to current xFP tags. Peptides will be made membrane permeant with the tat sequence or by myristoylation. This transformative approach will allow detection of proteins within minutes in living systems circumventing technically complicated, time consuming, and expensive genetic protein tagging. It will also dramatically accelerate (by 50fold) protein distribution in fixed cells by high and super-resolution microscop as it will not require the use of primary and secondary antibodies, not even washing steps. FMR peptides can be easily re-synthesized eliminating the variability inherent to antibody probes. Peptides will be reiteratively optimized to achive high affinity and specificity. My long-standing and overarching interest is to determine the molecular mechanisms that govern postsynaptic function (e.g., Science 293,98; Science 293,2205; Science Signaling 10, eaaf9659; Nature 411,801; Neuron 74,1023; Neuron 78,483; Neuron 81,249; Neuron 88,528; Neuron 97, 1094; Neuron 98, 783; EMBO J. 26,4879; EMBO J. 29,482; EMBO J. 31,1203; EMBO J. 33,1341; EMBO J. 35,1330; EMBO J. 36,1330; EMBO J. 37, 122). PSD-95 determines postsynaptic localization of glutamate receptors. Development of FMR-peptides that fluoresce upon specific binding to these proteins will allow live imaging of their localization and of the dynamic changes synapses with those proteins undergo over time at resting and stimulated conditions in cultured neurons and ultimately in the brain in vivo. Ultimately, I envision to develop FMR-peptides for >100 pre- and postsynaptic proteins. Others will apply our technology inside and outside the CNS in all fields of biomedical research. Page 1